Astrocytes are a major class of non-neuronal cells in the brain whose crosstalk with neurons at the synaptic and circuit levels remains poorly understood. While in vivo two-photon microscopy has revealed spatiotemporally diverse astrocytic signatures of intracellular Ca2+ transients, the scarcity of tools that manipulate the genetic makeup and physiological activity of astrocytes with spatial and temporal precision in vivo has restricted investigation of their physiological impact on neurons to predominantly correlational studies. Here, we propose developing three novel and mutually independent tools that target three crucial functions of astrocytes: gene expression, intracellular signal transduction, and glutamate uptake. In Aim 1, we will develop a CRISPR/Cas9- based platform to simultaneously knockout multiple genes selectively in astrocytes. Current mouse astrocytic gene ablation studies rely on a small number of Cre-LoxP recombinase transgenic lines, which target only a single gene and often lack temporal and spatial control. We propose creating a novel astrocyte-specific, temporally inducible, CRISPR/Cas9 conditional transgenic mouse model with an innovative viral platform for ablating multiple genes using a single virus Multi-gRNA, Cys4-mediated, Universal Targeting System (MRCUTS). We will apply this system in cultured astrocytes to target the Itpr2 and Adra1a/b genes (aim 1a), validate the tool and compare its efficacy to current Cre-LoxP methods (aim 1b), and probe a new functional role of astrocytes in arousal by using MRCUTS to simultaneously ablate two subtypes of noradrenergic receptors (Adra1a/b) (aim 1c). In Aim 2, we will develop a method for optogenetically activating G-protein signaling cascades in astrocytes. Current methods for modulating astrocyte signaling, such as DREADDs, lack temporal precision. We will develop and characterize the use of optogenetically activated G-protein receptors (opto-XR) in astrocytes to probe astrocyte signal transduction on physiologically-relevant timescales, first in vitro (aim 2a), then in vivo using 2-photon microscopy to measure astrocyte calcium dynamics (aim 2b), and subsequently explore the effects of astrocytic G-protein signal transduction on neuronal physiology using opto-XR in conjunction with astrocyte-neuron dual-calcium imaging (aim 2c). In Aim 3, we will develop an in vivo method for optogenetically disrupting glutamate uptake by astrocytes. Screening for mutations in ChR2, and combining four mutations, results in a light-gated ion channel, ChromeQ that possesses order-of-magnitude reductions in calcium and proton conductance while increasing sodium currents. We will record from astrocytes in acute brain slices to parameterize optogenetically activated sodium currents and determine effects on both astrocyte transporter currents and nearby neurons (aim 3a), examine how disrupting glutamate uptake via chromeQ affects astrocyte calcium dynamics and neuronal response properties in vivo (aim 3b), and explore the effects of ChromeQ on neuronal physiology and motor learning (aim 3c). The tools proposed here will enable a deeper understanding of astrocyte-neuron crosstalk in normal brain function and its disruption in brain disorders.